1. Technical Field
This invention relates to peptide ligands from viruses, e.g., human cytomegalovirus (HCMV) with improved immunogenicity wide recognition among HLA subtypes and methods for their production. These peptides are able to activate cytotoxic T lymphocytes at extremely low concentrations and therefore are suitable for use as vaccines.
2. Description of the Background Art
The HCMV genome is relatively large and has the capacity to encode more than 200 proteins. HCMV is composed of a nuclear complex of double-stranded DNA surrounded by capsid proteins having structural or enzymatic functions, and an external glycopeptide and glycolipid-containing membrane envelope. HCMV is a member of the Herpes virus family and has been associated with a number of clinical syndromes.
HCMV infection is relatively common and is usually self-limiting in the healthy, immunocompetent child or adult. However, approximately 10% of all newborn infants carry HCMV; the virus can cause severe congenital disease in the fetus or infant. For example, HCMV is a common cause of mental retardation in children who acquire the infection in utero from mothers carrying an active infection. Some of these newborn infants suffer congenital birth defects; others carry cytomegalovirus for some time before they show symptoms of the disease.
Other syndromes have been noted in persons carrying a persistent and apparently asymptomatic HCMV infection, for example, restenosis. HCMV is also associated with morbidity and mortality in immuno-compromised patients, such as patients suffering from acquired immunodeficiency syndrome (AIDS). AIDS patients infected with HCMV often suffer impairment of some of their vital organs, including the salivary glands, brain, kidney, liver and lungs as a result of the effects of HCMV disease. Furthermore, HCMV is associated with a wide spectrum of classical syndromes including mononucleosis and interstitial pneumonia. HCMV also has oncogenic potential and possible association with certain types of malignancies, including Kaposi""s sarcoma.
Because human cytomegalovirus is relatively common, yet is associated with some extremely serious health conditions, considerable effort has been made to study the biology of the virus with the aims of improving diagnosis and developing preventive and therapeutic strategies.
Viral infection of a host stimulates the processing of viral proteins through the Class I pathway, resulting in antigenic peptides which are presented by antigen presenting cells in the context of cell-surface MHC Class I. A CD8+CTL response is an important part of a mammalian host""s response to certain acute viral infections. Differentiation of CD8+T cells into mature CTL generally leads to clearance or control of the viral infection. The observations that HCMV infection is wide-spread and persistent and can become reactivated and clinically important in the immunosuppressed patient, suggest that virus-specific T cells, including HCMV-specific CTL, play an important role in both the control of persistent infection and recovery from HCMV disease. In a CD8+CTL response, a processed form of a viral protein (a minimal cytotoxic epitope) is recognized by CD8+CTL in combination with MHC Class I molecules. A minimal cytotoxic epitope of 8-12 amino acids can prime an antigen presenting cell to be lysed by CD8+CTL, as long as the correct MHC molecule is expressed on its surface.
Certain viral structural proteins which exist in large quantity in the viral particle, such as pp65 in HCMV, are chaperoned into infected host cells early in infection. Structural virion proteins are immuno-dominant target antigens important in the production of HCMV-specific CTL responses. The pp65 protein has been identified as a target antigen which is present in the peripheral blood of most asymptomatic HCMV seropositive individuals. McLaughlin-Taylor et al., J. Med. Virol. 43:103-110 (1994). The pp65495-503 CTL epitope (SEQ ID NO: 1; NLVPMVATV) from HCMV is universally recognized among HCMV-seropositive individuals who express HLA A*0201. Moreover, CTL against pp65495-503 recognize and lyse HCMV infected cells in vitro within an hour of infection. Thus, these CTL which recognize pp65495-503 may be important for limiting HCMV reactivation and progression of HCMV disease. The ability to induce a cellular immune response to pp65 therefore is extremely important in protecting both immuno-compromised and normal individuals from HCMV disease. However, the binding affinity of the pp65495-503 epitope to HLA A*0201 is only moderately strong.
One method of eliciting virus-specific CTL is to immunize with a vaccine peptide representing a minimal cytotoxic epitope defined-for a viral antigen in the context of a particular MHC restriction element. Such a vaccine boosts the CTL memory response to the virus in individuals carrying that MHC restriction element. Vaccine developers have become increasingly interested in immunizing with minimal cytotoxic epitopes rather than virus proteins because they can bind to MHC Class I molecules in the host through direct binding of the cell surface molecules without internalization or processing.
Individual MHC Class I molecules preferentially bind peptides of a given motif. The amino acid sequence of specific positions of the motif known as xe2x80x9canchor positionsxe2x80x9d are invariant. Falk et al., Nature 351:290-296 (1991). Amino acid positions other than the anchor position also contribute to a lesser degree to the specificity of peptide binding to MHC Class I molecules. Additionally, residues at positions within the CTL epitope which do not interact with MHC may interact with T cells. Some amino acid residues of the epitope contact the T cell receptor of the responding T cell, some contact the MHC restricting allele expressed on an antigen presenting cell, and some do not strongly contact either. Fremont et al., Science 257:919-927 (1992); Madden et al., Cell 75:693-708 (1993); Ono et al., J. Immunol. 161:5454-5463 (1998). The binding of amino acid residues to MHC or T cell receptor structures is independently governed, so that substitution of T cell receptor binding amino acid residues in some cases will not interfere with the binding to the MHC molecule on the surface of an antigen presenting cell. Sette et al., Mol. Immunol. 31:813-822 (1994); Vierboom et al., J. Immunother. 21:399-408 (1998); Valmori et al., J. Immunol. 161:6956-6962 (1998).
Peptides from the melanoma-specific antigen gp-100 have been altered at the anchor positions for increased binding to HLA A*0201, which resulted in the increased efficiency of stimulation of CTL from melanoma patients. Parkhurst et al., J. Immunol. 157:2539-2548 (1996). Similarly, CTL epitopes specific for murine H-2 Kd, Kb and Db were substituted at anchor positions, resulting in enhanced immunogenicity. Vierboom et al., J. Immunother. 21:399-408 (1998). On the other hand, directed substitution at anchor positions did not produce ligands that stimulated high-affinity CTL directed toward cancer antigens which were able to cause tumor regression. Clay et al., J. Immunol. 162:1749-1755 (1999). Therefore, different approaches need to be considered in defining residue substitutions that might influence the immunogenicity and vaccine properties of the pp65495-503 CTL epitope. The complexity of the molecular interactions between the individual molecular components of the immune system complex (TCR, peptide and MHC) has thwarted most attempts at significantly enhancing the binding affinity and immunogenicity of most peptide MHC ligands. Clay et al., J. Immunol. 162:1749-1755 (1999); Parkhurst et al., J. Immunol. 157:2539-2548 (1996); Leggatt et al., J. Immunol. 161:4728-4735 (1998).
In most cases, CTL epitopes are between 8-11 amino acids long as determined by either mass spectrometry or Edman degradation of peptides eluted from MHC molecules. Peptides which bind to MHC molecules after infection with virus are referred to as xe2x80x9cnaturally processed epitopes.xe2x80x9d These peptides require no further proteolytic processing to sensitize the transporter for antigen processing (TAP) deficient cell line, T2, for CTL-mediated lysis. Synthetic peptides having the sequence of these minimal length peptides (and certain variants) also can bind to MHC molecules and sensitize targets for lysis by CD8+CTL. Rammensee et al., Annu. Rev. Immunol. 11:213-244 (1993). More importantly, these peptides are capable of eliciting CTL that control or clear viral infections. Del et al., J. Virol. 65:3641-3646 (1991); Schulz et al., Proc. Natl. Acad. Sci. USA 88:991-993 (1991). An important objective therefore is to develop vaccination strategies that will successfully combat disease utilizing CTL epitopes or their analogs.
Accordingly, this invention comprises a method of producing a human cytomegalovirus peptide cytotoxic T lymphocyte epitope analog with improved immunogenic potency and retained breadth of recognition of MHC allele subtypes relative to the native human cytomegalovirus peptide cytotoxic T lymphocyte epitope which comprises providing a sequence of a human cytomegalovirus peptide cytotoxic T lymphocyte epitope and a combinatorial peptide library which contains peptide analogs of said viral peptide cytotoxic T lymphocyte epitope; screening said combinatorial peptide library for immunogenicity; analyzing the results of said screening to select one or more peptide analogs which may have improved immunogenicity relative to the native human cytomegalovirus peptide cytotoxic T lymphocyte epitope; assaying said one or more peptide analogs for immunogenicity; analyzing the results of the assay in step (d); selecting one or more highly immunogenic peptide analogs based on the analysis of step (e); assaying said highly immunogenic peptide analogs for broad recognition of MHC allele subtypes; analyzing the results of the assay in step (g); selecting one or more highly immunogenic peptide analogs with retained breadth of recognition of MHC allele subtypes relative to the native human cytomegalovirus peptide cytotoxic T lymphocyte epitope based on the analysis of the earlier steps; and synthesizing the peptides selected in step (i). In another embodiment, the invention provides a method which further comprises, before synthesizing the peptides, creating a library of peptide analogs from the highly immunogenic peptide analogs of step (f) containing substitutions of the native viral peptide cytotoxic T lymphocyte epitope; assaying said library of peptide analogs containing substitutions of the native viral peptide cytotoxic T lymphocyte epitope for both immunogenicity and breadth of recognition of MHC allele subtypes; and analyzing the results of the assays in step (B).
In further embodiments, the invention provides cytotoxic T lymphocyte epitope analogs produced according to the methods described above, for example, SEQ ID NOS: 38, 52 and 64. In further embodiments, the invention provides vaccines comprising such peptides.